The invention relates to the production and application of protozoa cultures of Histomonas meleagridis (H. meleagridis).
The flagellated protozoan Histomonas meleagridis is responsible for histomonosis (syn. blackhead disease) in poultry, a disease that occurs mainly in turkeys and chickens. The characteristics of the disease are necrotic lesions in the liver, thickening and ulceration of the caecal wall and sulphur-coloured droppings. Histomonosis causes high mortalities, especially in flocks of turkeys. Chickens show a higher resistance to histomonosis and lesions are usually confined to the caeca (McDougald, Avian Dis. 49 (2005), 462-476; Springer, Exp. Parasitol. 28 (1970), 383-392).
Following the ban of effective pharmaceuticals and food additives, which were licensed against flagellates in most countries in Europe and North America, the scientific interest in H. meleagridis has increased in the past decade due to the threat it poses to poultry flocks and because of the great financial losses associated with outbreaks of the disease.
The parasite H. meleagridis belongs to the order Trichomonadida, family Dientamoebidae. Common features of the protozoan include the parabasal apparatus with one flagellum, hydrogenosomes and feed vacuoles with starch granules or bacteria. Such features underline the high importance of interactions between bacteria and the protozoan parasite, both in vitro and in vivo (Delappe et al., Exp. Parasitol. 2 (1953), 79-86).
Goedbloed et al. (Avian Dis. 6 (1962), 302-315) disclosed the addition of bacteria to Histomonas cultures, wherein liver extracts from a turkey have been supplemented with E. coli to produce a monoxenic culture of H. meleagridis (“monoxenic” meaning that a single exogenous bacterial culture is added to the biopsy material which contains all the naturally present bacteria in the liver material, such as E. coli or cocci. Such monoxenic cultures have been used for establishing cultures of H. meleagridis, however, the presence of a bacterial biotope was always held necessary to establish such cultures (EP 1 721 965 A); it follows that for establishing a H. meleagridis culture, presence of a bacterial component is essential.
In contrast to the report of Goedbloed et al., studies on the in vitro cultivation of H. meleagridis, obtained from existing cultures containing a mixed turkey caecal bacterial flora, together with live and killed cells of E. coli or Escherichia freundii demonstrating that single bacterial strains are not suitable for the continuous in vitro propagation of the protozoan (Lesser, Helminthol. Soc. Wash. 31 (1964), 265-266). Consequently, the ability of monobacterial cultures to support the growth of the parasite in vitro has recently been questioned (Hauck et al., J. Parasitol. 96 (2010), 1-7). The issue is extremely important for the in vitro cultures currently under study, all of which contain the wildtype caecal bacterial flora of the birds from which H. meleagridis was isolated (e.g.: van der Heijden et al., Avian Pathol. 34 (2005), 505-508). The establishment of clonal protozoan cultures from the faeces of a diseased turkey offers new opportunities for the continuous and extended examination of the interactions between protozoa and bacteria (EP 1 721 965 A). Such clonal cultures would be a good start for establishing well defined cultures containing only a single bacterial strain and perhaps also enabling a specific exchange of bacteria.
On the other hand, vaccines against infections with H. meleagridis require safe antigens (Lund et al., Exp. Parasitol. 18 (1966), 403-407). A method for providing safe antigens is to provide attenuated cultures for vaccination. Attenuated cultures of H. meleagridis have been made available recently (Liebhart et al., Avian Pathol. 39 (2010), 399-403; Liebhart et al., Poultry Sci. 90 (2011), 996-1003; Hess et al., Vaccine 26 (2008), 4187-4193). However, such cultures are still derived from natural sources e.g. by individualisation using micro-manipulation (Hess et al., Parasitol. 133 (2006), 547-554; (more general:) Clark et al., Clin. Microb. Rev. 15 (2002), 329-341) and therefore still contain the bacterial flora of the natural sources. It has also recently made possible to provide clonal cultures of H. meleagridis (EP 1 721 965 A), however, although these cultures are clonal with respect to Histomonas, even these clonal cultures still contain a mixed bacterial culture of only roughly defined nature.
Market authorizations for vaccines, also in the veterinary field, require the defined composition of the effective components. It is therefore usually not possible to register and use cultures of undefined bacterial composition, including monoxenic cultures (as reported by Goedbloed et al., 1962), for industrial applications. It is therefore necessary to provide pharmaceutical compositions which do not only contain attenuated H. meleagridis cells but also are homogeneously designed with respect to the bacterial component.